The Dopamine Receptor of the Anterior Pituitary Gland - ACS

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The Dopamine Receptor of the Anterior Pituitary Gland Ligand Binding and Solubilization Studies M . G . C A R O N , B. F . K I L P A T R I C K , and A .

DE

LEAN1

Duke University Medical Center and Howard Hughes Medical Institute Research Laboratories, Departments of Physiology and Medicine, Durham, NC 27710

In the anterior pituitary gland dopamine inhibits the release of prolactin. The receptor for dopamine in the porcine anterior pituitary gland has been studied by direct ligand binding using [3H]n-propylapomorphine, an agonist or [3H]spiroperidol, an antagonist. The membrane -bound receptor appears to exist in two different forms which are reciprocally favored by agonists and antagonists. K values of agonists for these two forms of the receptor differ by 30-200 fold for various agonists but only 2- to 10-fold for [3H]spiroperidol. Guanine nucleotides (GTP; EC50 = 20-30 μΜ) convert the form of the receptor having high a f f i n i t y for agonists and low a f f i n i t y for antagonists almost completely to a form having low a f f i n i t y for agonists and high a f f i n i t y for antagonists. This effect of guanine nucleotides can be mimicked qualitatively and quantitatively by treatment of membranes with either N-ethylmaleimide (EC50 = 6 μΜ) or heat (53°for 4 min.) without affecting the total number of receptors as measured by [3H]spiro peridol binding. Treatment of membrane preparations with the detergent digitonin (1-2%) leads to the solubilization of up to 30% of the total receptor sites with retention of their ability to bind ligands with a dopaminergic s p e c i f i c i t y . S o l u b i l i z a t i o n , however, results in a loss of the nucleotide s e n s i t i v i t y of the receptor. Labelling of membranes with [3H]n-propylapomorphine prior to solubilization D

1

Current address: Clinical Research Institute of Montreal, 110 West Pine Avenue, Montreal, Quebec H2W 1R7, Canada 0097-6156/83/0224-0073$06.00/0 © 1983 American Chemical Society In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

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yields a s o l u b l e a g o n i s t - r e c e p t o r complex whose affinity can be modified by guanine n u c l e o t i d e s . These r e s u l t s suggest the involvement o f another component, presumably a guanine n u c l e o t i d e binding p r o t e i n , i n the mechanism of i n t e r a c t i o n of dopaminergic ligands with the dopamine receptor of the porcine a n t e r i o r pituitary gland.

The d i v e r s e p h y s i o l o g i c a l e f f e c t s o f catecholamines i n mammalian t i s s u e s are mediated by s p e c i f i c r e c e p t o r s . The e f f e c t o f epinephrine and norepinephrine are mediated by betaand alpha-adrenergic receptors w h i l e those o f dopamine are e l i c i t e d by d i s t i n c t receptor s i t e s . In the l a s t decade, dopamine receptors have been i d e n t i f i e d as being involved i n the modulation o f the enzyme adenylate c y c l a s e . The o r i g i n a l observations o f Kebabian e t a l . (I) t h a t a dopamine stimulated adenylate c y c l a s e was present i n the c e n t r a l nervous system was one of the f i r s t i n d i c a t i o n s as t o a p o s s i b l e biochemical mediator f o r dopaminergic responses. More r e c e n t l y , several reports have suggested that a pharmacologically d i s t i n c t subtype o f dopamine receptor might be coupled to an a t t e n u a t i o n of the e f f e c t o r , adenylate c y c l a s e , i n some t i s s u e s (2^3,4·). The c l a s s i f i c a t i o n proposed by Kebabian and Calne (5J oT two d i f f e r e n t subtypes termed D. and D« was based on pharmacological d i f f e r e n c e s between s i t e s i d e n t i f i e d by l i g a n d binding and s i t e s mediating dopamine s t i m u l a t i o n of adenylate c y c l a s e . Whereas a t D, receptor s i t e s dopamine acts a t micromolar concentrations and butyrophenone n e u r o l e p t i c s are weak antagonists (μΜ), a t D~ receptor s i t e s dopamine i s more potent and butyrophenone n e u r o l e p t i c s are a c t i v e i n the nanomolar range. These l a t t e r pharmacological p r o p e r t i e s are observed i n the few systems where dopamine has been found t o i n h i b i t adenylate c y c l a s e . However, a more d e t a i l e d and complex c l a s s i f i c a t i o n c o n t a i n i n g up t o f o u r d i f f e r e n t subtypes (D..-D-) has been r e c e n t l y proposed (6,_7). This c l a s s i f i c a t i o n which i s based on d i f f e r e n t i a l l a b e l l i n g o f s i t e s with various agonist and antagonist ligands as w e l l as pharmacological and other d i f f e r e n c e s between these s i t e s has been reviewed by Seeman (8). In the a n t e r i o r p i t u i t a r y gland where dopamine regulates the s e c r e t i o n o f p r o l a c t i n release we i d e n t i f i e d dopaminergic binding s i t e s with the s p e c i f i c i t y o f the Dp subtype of receptor by d i r e c t l i g a n d binding of the ergot a l k a l o i d [ H]dihydroergocryptine [9). Using t h i s technique, i t was p o s s i b l e f o r the f i r s t time t o c o r r e l a t e the pharmacological p r o p e r t i e s o f these s i t e s with a d i r e c t p h y s i o l o g i c a l e f f e c t , the i n h i b i t i o n of p r o l a c t i n release (9>). More r e c e n t l y , i t has been p o s s i b l e t o c o r r e l a t e , i n the p i t u i t a r y gland, occupancy

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.


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of the receptor with i n h i b i t i o n of the enzyme adenylate c y c l a s e a c t i v i t y (10) o r c y c l i c AMP l e v e l s (11). In the b r a i n where these s i t e s had been i d e n t i f i e d p r e v i o u s l y using n e u r o l e p t i c s no such c o r r e l a t i o n had been p o s s i b l e . Because o f the complex r e l a t i o n s h i p between the binding o f the numerous agonist and antagonist l i g a n d s f o r the dopamine receptor and the d i f f e r e n c e s i n c h a r a c t e r i s t i c s and numbers o f the s i t e s l a b e l l e d by these l i g a n d s , we set out t o examine the p r o p e r t i e s o f the dopamine receptor o f the porcine a n t e r i o r p i t u i t a r y gland using both an agonist r a d i o l i g a n d , [ H]n-propylapomorphine, and an a n t a g o n i s t , [ H j s p i r o p e r i d o l . Q u a n t i t a t i v e a n a l y s i s o f these data suggest t h a t both ligands i n t e r a c t with a s i n g l e dopamine receptor i n the a n t e r i o r p i t u i t a r y gland. This r e c e p t o r , however, appears t o e x i s t i n two d i s t i n c t a f f i n i t y forms r e c i p r o c a l l y favored by agonists and a n t a g o n i s t s . One o f the two forms o f the r e c e p t o r , the agonist high a f f i n i t y / a n t a g o n i s t low a f f i n i t y form may r e s u l t from the s t a b l e a s s o c i a t i o n o f the receptor with a guanine n u c l e o t i d e binding p r o t e i n . Furthermore, t h i s form o f the receptor can be s o l u b i l i z e d with d i g i t o n i n ; however only upon p r e l a b e l l i n g o f the receptor s i t e with the l a b e l l e d agonist can n u c l e o t i d e - s e n s i t i v i t y be returned. This paper w i l l summarize the evidence supporting the f o r m u l a t i o n that a s i n g l e dopamine receptor l a b e l l e d by [ H ] s p i r o p e r i d o l e x i s t s i n two d i s t i n c t a f f i n i t y forms i n porcine a n t e r i o r p i t u i t a r y gland (12,13). This f o r m u l a t i o n appears as an a t t r a c t i v e a l t e r n a t i v e to the proposal o f Seeman and coworkers (8) o f two d i s t i n c t subtypes designated and f o r t h i s receptor. Q u a n t i t a t i v e R e l a t i o n s h i p o f Agonist and Antagonist Ligand Binding t o Porcine A n t e r i o r P i t u i t a r y Gland Membranes In p a r t i c u l a t e preparations o f porcine a n t e r i o r p i t u i t a r y gland prepared by d i f f e r e n t i a l c e n t r i f u g a t i o g (12), the agonist [ H]n-propylapomorphine and the antagonist [ H ] s p i r o p e r i d o l bind with the same appropriate dopaminergic s p e c i f i c i t y (Figure 1A and B) ( i . e . η-propylapomorphine^ apomorphine > ADTN > dopamine) (ADTN not shown f o r the [ H]NPA curves). I t should be noted that the agonist competition curves f o r [ H ] s p i r o p e r i d o l binding are complex o r m u l t i p h a s i c with slope f a c t o r s o f l e s s than 1. Q u a n t i t a t i v e a n a l y s i s o f these data by l i n e a r l e a s t square f i t t i n g (_14) i n d i c a t e t h a t the agonists compete f o r the s i t e s l a b e l l e d by [ H ] s p i r o p e r i d o l with two d i f f e r e n t a f f i n i t i e s ; roughly 50% o f the t o t a l [ H ] s p i r o p e r i d o l s i t e s e x h i b i t high a f f i n i t y f o r the agonists and the remaining s i t e s have lower a f f i n i t y f o r the same a g o n i s t s . The r a t i o o f the two agonist a f f i n i t i e s (Κ,,/Κ,) vary from 30 i n the case o f apomorphine (Κ = 4.7 ± 2.4 nM, μ


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K. = 160 ± 61 nM) t o 200 f o r η-propylapomorphine (Κ = 0 . 0 8 1 ± 0.022 ήΜ, K, = 18 ± 3 nM). On the other hand, agonist competition curves f o r [ H]n-propylapomorphine are monophasic with slope f a c t o r s around u n i t y (Figure IB) suggesting t h a t both the l a b e l l e d agonist and the competing agonist i n t e r a c t with a s i n g l e form of the r e c e p t o r . The IC values c a l c u l a t e d from the agonist competition o f [ H]n-propylapomorphine correspond c l o s e l y t o those obtained from the higher o f the two a f f i n i t y forms evidenced i n the agonist competition curves o f [ H ] s p i r o p e r i d o l (e.g. f o r n-propylapomorphine: K „ , 3 = 0.081 ± 0.022 nM vs. K , 3 " ! A ™ ^ ^ ± 0.022 nM). J -P °Py' PO P ) These data suggest t h a t whereas [ H ] s p i r o p e r i d o l l a b e l s the e n t i r e population o f the dopamine r e c e p t o r , only a p o r t i o n of these same s i t e s are l a b e l l e d with [ H]n-propylapomorphine. This i s confirmed by f i n d i n g s from s a t u r a t i o n isotherms which show that i n a given membrane preparation [ H]n-propylapomorphine l a b e l s with high a f f i n i t y ( K = 0.26 nM) only one-half as many s i t e s as [ H ] s p i r o p e r i d o l . The remaining [ H ] s p i r o p e r i d o l s i t e s possess a f f i n i t y that i s too low f o r agonists ( K = 18-20 nM f o r n-prooylapomorphine) t o be l a b e l l e d by d i r e c t binding with [ H]n-propylapomorphine with the concentrations of l i g a n d normally used t o perform s a t u r a t i o n isotherms (10 DM to 1 nM). Therefore, i t appears t h a t by d i r e c t b i n d i n g , [H]n-propylapomorphine l a b e l s only the agonist high a f f i n i t y form o f the receptor population l a b e l l e d by [ H ] s p i r o p e r i d o l . μ

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Guanine n u c l e o t i d e s have been i m p l i c a t e d i n several receptor systems as important modulators o f the a f f i n i t y o f receptors f o r t h e i r s p e c i f i c hormones o r agonists (_15). The e f f e c t s are u s u a l l y observed as a decrease i n the a f f i n i t y o f the receptor f o r agonists (_16). For example, i n the beta-adrenergic receptor system, guanine n u c l e o t i d e s have been shown t o reduce the a b i l i t y o f agonists t o compete f o r antagonist binding t o the receptor ( J 7 ) . In the betaadrenergic system, agonists promote the formation of a t e r n a r y complex (18) composed o f the hormone, receptor and a n u c l e o t i d e binding p r o t e i n (19). This ternary complex i s d e s t a b i l i z e d or i t s formation inhiïïited i n the presence o f guanine n u c l e o t i d e s (20). In dopaminergic receptor systems, s i m i l a r e f f e c t s o f guanine n u c l e o t i d e s on agonist potency a t the receptor have been reported (reviewed i n (8) and c f . r e f s . (19-24) c i t e d i n ( 1 2 ) ) . In order t o probe the r e l a t i o n s h i p o f the two d i f f e r e n t a f f i n i t y forms o f the dopamine r e c e p t o r , i t was o f i n t e r e s t t o


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Competition curves of a series of dopaminergic agonists for the direct binding of [ H] agonist and antagonist ligands. 3

Porcine anterior pituitary gland membranes prepared as described previously (12), were incubated with [ H]spiroperidol (~ 140 pM) (A) or [ H]n-propylapomorphine (~ 150 pM) (B) and increasing concentrations of the various agonists as shown. Membranes suspended in 50 mM Tris-HCl, 6 mM MgCh, 1 mM EDTA, 100 mM NaCl, 0.1% ascorbate, 10 μΜ pargyline, pH 7.4, at 25 °C were incubated for 60 min at 25 °C in a total volume of 1 mL containing 0.3 mg and 0.6 mg of membrane protein/assay for [ H]spiroperidol and [ H]npropylapomorphine, respectively. Binding was initiated by the addition of membranes, the incubations were terminated by adding 5 mL of cold 25 mM Tris-HCl, 2 mM MgCU (pH 7.4) (4 °C) and rapid vacuum filtration on GF/C or GF/B glass fiber filters with four additional 5 mL washings. Each agonist drug competed for binding to the level of nonspecific binding that was determined in the presence of 1 μΜ (-\-)butaclamol. Under routine conditions specific binding accounted for 80-90% of total binding for [ H]spiroperidol and 50-70% for [ H]n-propylapomorphine. In the experiment shown, 100% [ Η]spiroperidol binding corre sponded to 32 pM or ~ 107 fmol/mg whereas 100% [ H]n-propylapomorphine was 33 pM or 55 fmol/mg. The points represent experimental data, and the lines represent the best fit of the data according to a model for one or two binding sites analyzed as described previously (12). The experiment shown was performed in duplicates and is representative of three (A) and two (B) such experiments. (Reproduced with permission from Ref. 12. Copyright 1982, American Society for Pharmacology and Experimental Therapeutics.) 3

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examine the e f f e c t of guanine nucleotides on dopaminergic l i g a n d binding to porcine a n t e r i o r p i t u i t a r y membranes. As shown i n Figure 2, the guanine nucleotide Gpp(NH)p markedly decreases the apparent potency of an agonist such as n-propylapomorphine i n competing f o r the binding of the antagonist [ H j s p i r o p e r i d o l . The c o n t r o l competition curve which i s b i p h a s i c and shallow i s s h i f t e d to lower apparent potency and steepened i n the presence of guanine n u c l e o t i d e s . In a d d i t i o n , as w i l l be discussed below, an apparent increase i n [ H j s p i r o p e r i d o l binding was observed i n the presence of guanine n u c l e o t i d e s . Q u a n t i t a t i v e a n a l y s i s of the c o n t r o l competition curve i n d i c a t e d that 50% of the [ H j s p i r o p e r i d o l s i t e s d i s p l a y high a f f i n i t y f o r the agonist (Km = 0.29 ± 0.13 nM) and 50% of the s i t e s d i s p l a y e d a lower a f f T n i t y (K. = 8.3 ± 2.0 nM). In the presence of n u c l e o t i d e , however, the proportion of the agonist high a f f i n i t y form i s reduced to only 10 to 15% with no s i g n i f i c a n t change i n a f f i n i t y ( K = 0.20 ± 0.35 nM). The remaining 85 to 90% of the s i t e s , then show low a f f i n i t y f o r the agonist i n d i s t i n g u i s h a b l e from the lower agonist a f f i n i t y of the c o n t r o l curve (8.3 ± 2.0 nM vs. 12.0 ± 1.9 nM). Thus, guanine nucleotides appear to decrease the proportion of the receptor e x h i b i t i n g high a f f i n i t y f o r agonists (12). This e f f e c t can be t e s t e d d i r e c t l y by examining the e f f e c t s of nucleotides on d i r e c t agonist b i n d i n g . As shown i n Figure 3 i n the presence of i n c r e a s i n g concentrations of GTP, there i s a progressive l o s s of the number of agonist high a f f i n i t y form of the receptor detectable by d i r e c t [ Hjnpropylapomorphine binding ( c o n t r o l = 26.7 ± 2.1 pM; + 10 yM GTP = 19.6 ± 2.4 pM and + 1 mM GTP = 6.0 ± 1.4 pM). The a f f i n i t y of the remaining s i t e s l a b e l l e d by [ H]n-propylapomorphine remains e s s e n t i a l l y the same f o r the l i g a n d . The e f f e c t of guanine nucleotides on t h i s system, much l i k e i n other receptor systems (18), i s to decrease the proportion of the receptor e x i s t i n g i n an agonist high a f f i n i t y form. This e f f e c t appears as e i t h e r an apparent decrease i n the a b i l i t y of agonists to compete f o r antagonist r a d i o l i g a n d binding or an actual decrease i n the number of s i t e s l a b e l l e d with high a f f i n i t y by a l a b e l l e d agonist. This l a t t e r e f f e c t i s due to the f a c t that the agonist lower a f f i n i t y form of the receptor u s u a l l y cannot be l a b e l l e d by d i r e c t agonist l i g a n d b i n d i n g . Thus, these r e s u l t s are c o n s i s t e n t with the t h e s i s that the dopamine receptor i n the porcine a n t e r i o r p i t u i t a r y gland can e x i s t i n two d i f f e r e n t a f f i n i t y forms w i t h respect to agonists and that these two s t a t e s can be i n t e r c o n v e r t e d by guanine n u c l e o t i d e s . These s p e c i f i c e f f e c t s of guanine n u c l e o t i d e s s t r o n g l y suggest that high a f f i n i t y i n t e r a c t i o n s of the receptor with agonists i n v o l v e another component which mediates the e f f e c t s of n u c l e o t i d e s . M

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Effect of the guanine nucleotide Gpp(NH)p on competition of the agonist n-propylapomorphine for [ H]spiroperidol binding. 3

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Membranes were incubated as described in Figure 1 with [ Hjspiroperidol (~ 160 pM) and increasing concentrations of n-propylapomorphine in the presence and absence of 100 μΜ Gpp(NH)p. The lines are the best fit obtained with a model for two affinity forms of the receptor as described previously (14). The experiment shown is representative of 10-12 experi ments. (Reproduced with permission from Ref. 12. Copyright 1982, American Society for Pharmacology and Experimental Therapeutics.)

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[ H] NPA ADDED (pM)

Figure 3. Effects of guanine nucleotides on the direct binding of apomorphine.

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[ Η]n-propyl

Porcine anterior pituitary membranes were incubated with increasing concentrations (10 pM900 pM) of [ H]n-propylapomorphine in the presence of 10 μΜ and 1 mM GTP and in the absence of GTP as the control. Nonspecific binding was determined in the presence of 1 μΜ (-\-)butaclamol and was the same with or without the addition of GTP. Data were analyzed as described in Figure 1. The number of sites labelled by [ H]n-propylapomorphine was 26.7 pM (absence), 19.6 pM in the presence of 10 μΜ GTP and 6.0 pM in the presence of 1 mM GTP and dissociation constants for the ligand were 280 and 210 pM. The experiment was performed in duplicate and is representative of three such experiments. 3

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Guanine Nucleotides Modulation Antagonist?

RECEPTORS

o f Receptor A f f i n i t y f o r

As can be observed from competition experiments o f η-propylapomorphine f o r [ H j s p i r o p e r i d o l binding (Figure 2 ) the presence o f guanine n u c l e o t i d e s y i e l d s an apparent increase i n the binding o f [ H j s p i r o p e r i d o l . This e f f e c t i s q u i t e d i f f e r e n t from the usual e f f e c t o f n u c l e o t i d e s observed on agonist binding (JL5,L8). However, e f f e c t s o f guanine n u c l e o t i d e s on antagonist binding have been described f o r muscarinic receptor systems (22^,23) and more r e c e n t l y f o r the beta-adrenergic receptor system Jzk). In order to explore f u r t h e r t h i s e f f e c t d e t a i l e d s a t u r a t i o n binding isotherms o f [ H j s p i r o p e r i d o l binding were performed i n the presence and absence o f a guanine n u c l e o t i d e . As shown i n Figure 4 , the c o n t r o l s a t u r a t i o n curve as represented i n the Scatchard p l o t coordinates i s s l i g h t l y c u r v i l i n e a r but becomes l i n e a r i n the presence o f 1 mM GTP. Q u a n t i t a t i v e a n a l y s i s by l i n e a r l e a s t square f i t t i n g i n d i c a t e s t h a t two K values (45 and 415 pM) can be significantly derived f o r [ Hjspiroperidol binding i n the absence o f GTP and a single value o f 56 pM (not s i g n i f i c a n t l y d i f f e r e n t than 45 pM) i n the presence o f GTP. The two a f f i n i t y forms which are present i n roughly equal proportions i n the c o n t r o l curve are converted to a s i n g l e high a f f i n i t y form i n the presence o f GTP w i t h no change i n the t o t a l number o f s i t e s f o r [ H j s p i r o p e r i d o l ( 2 5 ) . GTP d i s p l a y s the same dose response (EC™ = 32 yMj f o r i t s e f f e c t on L H j s p i r o p e r i d o l binding as the e r f e c t on agonist binding (EC = 17 yM). Thus, i t appears that the same two a f f i n i t y fortfis o f the receptor which are d i s c r i m i n a t e d by agonists may a l s o be d i s c r i m i n a t e d by antagonists although i n a r e c i p r o c a l f a s h i o n . The d i s c r i m i n a t i n g power o f [ H j s p i r o p e r i d o l f o r the two forms o f the receptor i s , however, much weaker than f o r agonists ( 2 - 1 0 f o l d f o r [ ^ s p i r o p e r i d o l and 30-200 f o l d f o r a g o n i s t s ) . Thus, i t would appear t h a t i n the presence o f n u c l e o t i d e s the agonist high a f f i n i t y form/antagonist low a f f i n i t y form i s converted t o the agonist low a f f i n i t y / antagonist high a f f i n i t y form of the receptor. The p o s s i b l e p h y s i o l o g i c a l relevance o f these f i n d i n g s w i l l be discussed l a t e r i n t h i s chapter. D

5 Q

N-ethylmaleimide and Heat Treatments of Membranes Mimic the E f f e c t o f Guanine Nucleotides on Ligand Binding A l l the data presented above point to the involvement of a p u t a t i v e guanine n u c l e o t i d e binding p r o t e i n i n the i n t e r a c t i o n of agonists with the dopamine receptor i n the porcine a n t e r i o r p i t u i t a r y gland. We, t h e r e f o r e , wanted to obtain more compelling evidence f o r the i n t e r a c t i o n o f a n u c l e o t i d e binding



CARON

Anterior

E T AL.

Ό

Pituitary

10

Gland's Dopamine

20

SPECIFIC [3H]SPIR0

Figure 4.

30

Receptor

40

BINDING (pM)

Effects of GTP on the binding isotherms of the antagonist peridol.

3

[ H]spiro

Porcine anterior pituitary membranes were incubated as described in Figure 1 with ^Hjspiro peridol (10-1100 pM). Nonspecific binding was determined in the presence of 1 μΜ (+)butaclamol. Data were analyzed by curve fitting with a model for the binding of the radioligand to one or two classes of sites (14). Data are presented in the Scatchard plot coordinates. The experiment was performed in duplicate and is similar to several other experiments with the same results but it represents an extreme case of the ability of the [ H] antagonist to discriminate between the two forms of the receptor. The two dissociation constants calculated for the control curve are different by about 10-fold. (Reproduced with permission from Ref. 25. Copyright 1982, American Society for Pharmacology and Experimental Therapeutics.) 3



82

DOPAMINE

RECEPTORS

e n t i t y with the receptor. N-ethylmaleimide (NEM), a s u l h y d r y l a l k y l a t i n g agent, mimics the e f f e c t s o f n u c l e o t i d e s on agonist binding a t the w e l l - c h a r a c t e r i z e d beta-adrenergic receptor o f the f r o g e r y t h r o c y t e (26). In a manner s i m i l a r t o the e f f e c t of guanine nucleotides i n t h a t system, NEM prevents the formation o f the agonist high a f f i n i t y form o f receptor with no change i n t o t a l number of receptors. Moreover, i t i s apparent that the guanine n u c l e o t i d e r e g u l a t o r y p r o t e i n a c t i v i t y of the beta-adrenergic receptor demonstrates e x q u i s i t e s e n s i t i v i t y to temperature. Ross e t a l . (27) showed that incubation o f S-49 lymphoma c e l l membranes a t 50° i n a c t i v a t e s the r e g u l a t o r y p r o t e i n a c t i v i t y with a t i o f 2-8 minutes as assayed by r e c o n s t i t u t i o n s t u d i e s . The r e g u l a t o r y p r o t e i n a c t i v i t y assayed by r e c o n s t i t u t i o n i s the same f u n c t i o n a l e n t i t y which i n t e r a c t s with the beta-adrenergic receptor t o form the high a f f i n i t y agonist ternary complex (19). Therefore, i t i s reasonable t o assume that NEM and heat treatment i n t e r f e r e with the r e c e p t o r - a f f i n i t y modulating f u n c t i o n of the guanine n u c l e o t i d e r e g u l a t o r y p r o t e i n i n t h a t system. Treatment o f porcine a n t e r i o r p i t u i t a r y gland membranes with e i t h e r NEM at concentrations between 1-100 yM o r heat (53° f o r 4 min.) q u a l i t a t i v e l y and q u a n t i t a t i v e l y mimics the e f f e c t s of guanine nucleotides on dopaminergic l i g a n d b i n d i n g . As shown i n Figures 5A and B- competition curves o f the agonist η-propylapomorphine f o r [ H j s p i r o p e r i d o l binding are s h i f t e d to the r i g h t and steepened. These e f f e c t s are i d e n t i c a l to the a b i l i t y of guanine nucleotides t o decrease the proportion of agonist high a f f i n i t y form of the receptor. S i m i l a r l y , i n c r e a s i n g concentrations of NEM from 1 yM t o 100 yM cause a progressive decrease i n the d i r e c t binding of the agonist [ Hjn-propylapomorphine (Γ3). Moreover, i t can be observed that both NEM and heat treatments seem t o a f f e c t [ H j s p i r o p e r i d o l binding i n a manner s i m i l a r to guanine n u c l e o t i d e s since an apparent increase i n binding of [ H j s p i r o p e r i d o l can be observed (Figures 5A and B). Heat treatment of membranes does not increase [ H j s p i r o p e r i d o l binding~as much as GTP because under these c o n d i t i o n s (53°for 4 min.) [ H j s p i r o p e r i d o l binding i s s l i g h t l y i n h i b i t e d (15-20%) (not shown) (13). Thus, these r e s u l t s suggest that the e f f e c t s o f heat and NEM treatments mimic the modulation o f dopaminergic l i g a n d binding by guanine nucleotides and are very s i m i l a r to t h e i r e f f e c t s on beta-adrenergic systems. The only exception appears to be the r e l a t i v e s e n s i t i v i t y of the dopaminergic system t o NEM (13,26). Whereas, on dopaminergic b i n d i n g , NEM e l i c i t s i t s e f f e c t s with an EC™ = 6 yM, concentration i n the m i l l i m o l a r range are required Vor the same e f f e c t s on beta-adrenergic agonist b i n d i n g . By analogy with the beta-adrenergic receptor system, these data support an i n t e r a c t i o n of a guanine


CARON

Anterior

E T A L .

Έ

25

Q

20

Pituitary

Gland's Dopamine

Receptor

83

CL


Ζ ZD Ο CD

15

Ο 01

10

CL CO

γ—ι Χ

,ΓΟ

,

5 0

12

II

10

9

8

7

6

- L O G [NPA] ( Μ )

60

S

50 _

Q

40

Ζ ZD Ο

ûû

Β 1

- É _ t •

30

Ο 01

20

CL CO

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1

10 0

1

1

12

II

1

1

1

10

9

8

7

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ι

6

5

- L O G [NPA] (M)

Figure 5. Comparison of the effects of NEM and heat treatments and the presence of GTP on the ability of n-propylapomorphine (agonist) to compete for the binding of the antagonist [ H]spiroperidol. 3

3

Membranes were incubated with [ H]spiroperidol (~ 109 pM) for 1 h at 25 ° C and increasing concentrations of n-propylapomorphine (A). Key to A: · , control membranes; • , membranes treated in the presence of 100 μΜ NEM as described below; and A, membranes incubated in the presence of 1 mM GTP. The curves represent computer drawn lines that best fit the data points. NEM treatment was performed as follows: Membranes were incubated with the indicated concentration of NEM for 30 min at 25 °C prior to addition of the radioligand. The same results were obtained whether or not the membranes were washed free of residual NEM prior to the binding assay. All three curves are from a single experiment that is representative of three experiments. Control membranes and membranes treated at 53 °C for 4 min as described below were incubated with [ Hjspiroperidol (190 pM) in the presence of increasing concen trations of n-propylapomorphine. One set of membranes were also incubated in the presence of 100 μΜ GTP. Data were analyzed by computer-based methods as described in Figure 1 with the lines representing the best fit to the data (14). The experiment was performed and is representative of two such experiments. (Reproduced with permission from Ref. 13. Copyright 1982, American Society for Pharmacology and Experimental Therapeutics.) 3


84

DOPAMINE

RECEPTORS

n u c l e o t i d e r e g u l a t o r y p r o t e i n with the dopamine receptor i n the a n t e r i o r p i t u i t a r y gland.


S o l u b i l i z a t i o n of the Dopamine Receptor One of the p r e r e q u i s i t e s f o r the p u r i f i c a t i o n and c h a r a c t e r i z a t i o n of a membrane-bound hormone receptor i s i t s s o l u b i l i z a t i o n i n an a c t i v e form, i . e . with r e t e n t i o n of the a b i l i t y of the receptor to i n t e r a c t with s p e c i f i c l i g a n d s . Several reports have appeared already documenting the s o l u b i l i z a t i o n of dopaminergic binding s i t e s from b r a i n t i s s u e (28-31) using detergents such as d i g i t o n i n and 3'-[(3 cholamidopropyldimethyl ami no)dimethyl-amonio]-1-propanesulfonate (CHAPS) or chaotropic agents. In the porcine a n t e r i o r p i t u i t a r y gland we report here that the dopamine receptor can be s o l u b i l i z e d using the detergent d i g i t o n i n . Treatment of membrane preparations with d i g i t o n i n (1-2%) i n 100 mM NaCl, 10 mM T r i s - H C l pH 7.4 releases 20-30% of the membrane-binding s i t e s i n a s o l u b l e form that cannot be sedimented at 100,000 xg f o r 60 min. S a t u r a t i o n isotherms of [ H j s p i r o p e r i d o l as measured by a Sephadex G-50 assay (32) f o r separation of bound from f r e e l i g a n d reveal a d i s s o c i a t i o n constant ( K ) of 0.5 nM f o r s p i r o p e r i d o l (not shown). Although, t n i s value i s somewhat lower than the K of 40-70 pM obtained f o r [ H j s p i r o p e r i d o l binding to p a r t i c u l a t e p r e p a r a t i o n s , the s p e c i f i c i t y of binding of the antagonist to the s o l u b i l i z e d s i t e s i s s i m i l a r to t h a t i n the membrane f r a c t i o n (n-propylapomorphine > ADTN > dopamine) (not shown) (12). As mentioned p r e v i o u s l y , agonist competition curves f o r [ H j s p i r o p e r i d o l binding i n p a r t i c u l a t e preparations are b i p h a s i c and high and low a f f i n i t y forms of the receptor f o r agonists can be documented; i n s o l u b i l i z e d preparations a s i n g l e low a f f i n i t y can be detected f o r the competition of agonists f o r s o l u b l e [ H j s p i r o p e r i d o l b i n d i n g . By comparison to the K values (0.080 and 18 nM) c a l c u l a t e d f o r n-propylapomorphine competition of p a r t i c u l a t e [ H j s p i r o p e r i d o l b i n d i n g , the value obtained from s o l u b i l i z e d preparations i s 35 nM. Moreover, the presence of guanine n u c l e o t i d e s i n the assays does not f u r t h e r reduce the a b i l i t y of the agonists to compete f o r [ H j s p i r o p e r i d o l binding (not shown). Therefore, i t appears t h a t s o l u b i l i z a t i o n i n t e r f e r e s with the a b i l i t y of agonists to i n t e r a c t with high a f f i n i t y with the receptor. This i s a common f i n d i n g i n several s o l u b i l i z e d receptor systems (33). D

D

D

However, i f the agonist high a f f i n i t y form of the receptor i s l a b e l l e d p r i o r to s o l u b i l i z a t i o n by i n c u b a t i o n of membranes with [ H]n-propylapomorphine then the agonist high a f f i n i t y form of the receptor appears to be s t a b l e to s o l u b i l i z a t i o n (Table I ) . Shown i n Table I are the r e s u l t s of three


3.

CARON

Anterior

ET AL.

Pituitary

Gland's Dopamine

Receptor

85


Table I S o l u b i l i z a t i o n by D i g i t o n i n o f Agonist and Antagonist P r e l a b e l l e d Dopamine Receptor from A n t e r i o r P i t u i t a r y Gland Membranes 3

[ H ] Ligand

Experiment

J

[ H]NPA fmol 63 55 35.9

% agonist high a f f i n i t y sites % 41 42 59

J

[ H]Spiro +1 nM NPA fmol 110 90

J

[ H]Spiro fmol 155 133 60

Membranes were incubated f o r 60 minutes a t 25° as described i n the legend t o Figure 1 w i t h 800 pM [ H]n-propylapomorphine (NPA) and 700 pM [ H ] s p i r o p e r i d o l ( S p i r o ) and [ H ] s p i r o p e r i d o l i n the presence o f 1 nM unlabel l e d NPA, a c o n c e n t r a t i o n s u f f i c i e n t t o saturate the agonist high a f f i n i t y form o f the receptor. A f t e r the i n c u b a t i o n , membranes were c e n t r i f u g e d and s o l u b i l i z e d by resuspending the membranes i n 1% d i g i t o n i n , 100 mM NaCl, 25 mM Tris-HCl 2 mM M g C l , 0.1% ascorbate pH 7.4 a t 4°C, s t i r r i n g on i c e f o r 30 minutes and c e n t r i f u g i n g a t 40,000 χ g f o r 45 minutes. S p e c i f i c binding f o r both ligands was determined by Sephadex G-50 chromatography o f the s o l u b i l i z e d samples from incubation with l i g a n d s alone o r l i g a n d s with 10" M (+)butaclamol. Results are expressed i n fmol o f s p e c i f i c binding i n 1 ml o f s o l u b i l i z e d preparation i n the r e s p e c t i v e experiments. % agonist high a f f i n i t y s i t e s i s expressed as the percent o f [ H3NPA s i t e s compared t o the number o f s i t e s l a b e l l e d with [ H ] s p i r o p e r i d o l . J

J

?



86

DOPAMINE

RECEPTORS

experiments i n which membrane preparations were~incubated with ["H]n-propylapomorphine, [ H j s p i r o p e r i d o l and [ H j s p i r o p e r i d o l i n the presence of 1 nM u n l a b e l l e d n-propylapomorphine p r i o r t o s o l u b i l i z a t i o n with d i g i t o n i n . Under these c o n d i t i o n s , the r e s u l t s i n d i c a t e t h a t about the same proportion o f agonist high a f f i n i t y form of the receptor can be s o l u b i l i z e d as that which can be evidenced i n membrane preparations ( i . e . about 50%). In the presence o f 1 nM n-propylapomorphine, a concentration s u f f i c i e n t to saturate the agonist high a f f i n i t y form of the receptor i n the membrane, the amount of [ H j s p i r o p e r i d o l binding s o l u b i l i z e d i s reduced by approximately the amount of [ H]n-propylapomorphine s i t e s s o l u b i l i z e d . In a d d i t i o n , the data i n Table I I i n d i c a t e that the s o l u b l i z e d agonist high a f f i n i t y form of the receptor remains s e n s i t i v e t o the e f f e c t of n u c l e o t i d e s . [ H]n-Propylapomorphine b i n d i n g , assayed i n the membranes i n the presence of Gpp(NH)p o r t r e a t e d with Gpp(NH)p a f t e r s o l u b i l i z a t i o n , i s reduced to about the same extent. As shown i n Figures 6A and B, t h i s apparent decrease i n the number of binding s i t e s f o r [H]n-propylapomorphine i n the presence of guanine n u c l e o t i d e s i n both membranes (Figure 6A) and s o l u b i l i z e d preparations (Figure 6B), can be a t t r i b u t e d to a decrease i n the a f f i n i t y o f the receptor f o r the a g o n i s t . This apparent decrease i n a f f i n i t y can be accounted by the increased d i s s o c i a t i o n rate o f [ H]n-propylapomorphine i n both preparations i n the presence of Gpp(NH)p. The r e s u l t s presented above are c o n s i s t e n t with the formulation postulated p r e v i o u s l y , that the receptor can e x i s t as two a f f i n i t y forms, one of which i s h i g h l y prefered or s t a b i l i z e d i n the presence of an agonist. In a d d i t i o n , these data i n d i c a t e that whereas the receptor can be s o l u b i l i z e d with apparent r e t e n t i o n o f i t s binding s p e c i f i c i t y , high a f f i n i t y i n t e r a c t i o n s of the receptor with agonists are not present i n s o l u b i l i z e d preparations unless s t a b i l i z e d p r i o r to s o l u b i l i z a t i o n by the presence o f an a g o n i s t . These r e s u l t s are e n t i r e l y analogous to the beta-adrenergic receptor system (34) where the component required f o r high a f f i n i t y agonist i n t e r a c t i o n s with the receptor has been documented to be a guanine n u c l e o t i d e binding p r o t e i n . Conclusions The d i s c r i m i n a t i o n i n the membrane preparations by agonists o f two a f f i n i t y forms o f the receptor allows the comparison of the potency o f these agonists with t h e i r a b i l i t y to e l i c i t i n h i b i t i o n p r o l a c t i n r e l e a s e . I t can be observed, that the K values of agonists observed f o r the high a f f i n i t y form of the receptor (Κ„) c o r r e l a t e very c l o s e l y with the a b i l i t y (EC™) o f these same agents to i n h i b i t p r o l a c t i n r e l e a s e (9) \ - 66 nM vs. E C = 35 Q

5

( d o p a m i n e )

5 Q

( d o p a m i n e )



3.

CARON

ET AL.

Anterior

Pituitary

Gland's Dopamine

Receptor

87

Table I I E f f e c t o f Guanine Nucleotides on High A f f i n i t y Agonist Binding to Membrane and S o l u b i l i z e d Receptor Preparations 3

Experiment 1 2

[ H]NPA s p e c i f i c binding (fmol) Membrane preparations S o l u b i l i z e d preparations Control +l00yMGpp(NH)p Control +100yMGpp(NH)p 193 132

19 (86)

40 44

8.6 (79) 13.0 (71)

Membranes were incubated as described i n Table I w i t h 800 pM [ H]n-propylapomorphine ( c o n t r o l ) and i n the presence o f 100 yM Gpp(NH)p. S p e c i f i c binding was determined as described i n the legend t o Figure 1. For data w i t h s o l u b i l i z e d p r e p a r a t i o n s , membranes were incubated with 800 pM [ H]n-propylapomorphine f o r 60 minutes a t 25°C then s o l u b i l i z e d as described i n Table I. The s o l u b i l i z e d samples were then incubated f o r 15 hours a t 4° i n the presence o r absence o f 100 yM Gpp(NH)p and s p e c i f i c binding determined by Sephadex G-50 chromatography from the d i f f e r e n c e between the above samples and samples which had been incubated with 10" M (+)butaclamol from the i n i t i a l i n c u b a t i o n . The numbers i n parenthesis i n d i c a t e the percent decrease i n agonist binding i n the presence o f Gpp(NH)p.



20

40

60

80

100

ι I 15

ι 20

Gpp(NH)p

Gpp(NH)p

ο

Gpp(NH)p

10

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80

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100 < °°

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(min)

30

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3

3

Effects of guanine nucleotides on the dissociation rate of [ Η]n-propylapomorphine binding from membrane-bound and solubilized receptor preparations.

TIME (min)

ι 10

Α

100/ιΜ

•

ΙΟ/ιΜ

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No

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5

40

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Gpp(NH)p

Membranes were incubated as described in Figure 1 with 500 pM [ H]n-propylapomorphine for 60 min at 25 °C to achieve equilibrium in the presence of 0.1% ascorbate (A). Dissociation was initiated by adding at time 0 an excess of (-{-)butaclamol (10~ M) or (-\-)butaclamol, plus 10 and 100 μΜ Gpp(NH)p. Samples were filtered on GF/B filters at time intervals shown. Nonspecific binding was determined by parallel incubations containing (-\-)butaclamol from the beginning of the incubation. The experiment performed in duplicate is representative of three such experiments. Membranes were also incubated as above with [ H]n-propylapomorphine to achieve equilibrium (B). After the incubation, membranes were centrifugea and resuspended in 1% digitonin 25 mM Tris-HCl, 100 mM NaCl, 2 mM MgCh, 0.1% ascorbate, pH 7.4, at 4 °C, stirred for 30 min on ice, and centri fugea at 40,000 χ g for 45 min to obtain the solubilized labelled receptor preparation. Dissociation of the ligand was started by adding at time 0 excess (-\-)butaclamol (10~ M) or (-\-)butaclamol and 100 μΜ Gpp(NH)p. Sam ples were incubated at 4 °C for the time intervals indicated, and binding was determined by Sephadex G-50 chromatography. The results are representative of two similar experiments.

Figure 6.

Ν

ο

Α


INDI


3.

CARON

ET A L .

Anterior

Pituitary

Gland's Dopamine

Receptor

89

nM). ions are much c l overa11 r e l a t i v e potency of an agonist f o r competing f o r l i g a n d binding i s compared with the p h y s i o l o g i c a l response (9). It is t h e r e f o r e reasonable t o p o s t u l a t e t h a t the p h y s i o l o g i c a l response t o dopamine, i . e . i n h i b i t i o n o f p r o l a c t i n r e l e a s e , i s a r e f l e c t i o n o f the high a f f i n i t y i n t e r a c t i o n o f dopaminergic agonists o r dopamine i t s e l f with the receptor i n the a n t e r i o r p i t u i t a r y gland. The d i s c r i m i n a t i o n o f the same two forms o f the receptor by antagonists i n a r e c i p r o c a l f a s h i o n t o agonists i s much more s u b t l e . F i r s t , the d i f f e r e n c e s i n a f f i n i t y observed f o r the antagonist [ H j s p i r o p e r i d o l are small ( 2 - 1 0 f o l d ) as compared to âgonits ( 3 0 - 2 0 0 f o l d ) , hence more d i f f i c u l t t o demonstrate unless extremely d e t a i l e d s a t u r a t i o n isotherms are performed. Moreover, the d i s c r i m i n a t i o n o f the two a f f i n i t y forms o f the receptor by an antagonist l i g a n d has been demonstrated f o r [ H j s p i r o p e r i d o l only as no other antagonists have been examined. The f a c t t h a t antagonists i n several receptor systems can s l i g h t l y d i s t i n g u i s h two forms o f a receptor probably has no major p h y s i o l o g i c a l s i g n i f i c a n c e w i t h respect to the a c t i o n s o f antagonists. Nonetheless, the q u a n t i t a t i v e aspect o f the binding o f [ H j s p i r o p e r i d o l t o the two forms o f the receptor and t h e i r modulation by guanine n u c l e o t i d e s can be taken as f u r t h e r support f o r the e x i s t e n c e o f these two d i f f e r e n t i n t e r c o n v e r t i b l e forms o f the receptor which appear more important i n the mechanism o f a c t i o n o f a g o n i s t s . In the beta adrenergic receptor system o f the f r o g e r y t h r o c y t e ( 1 8 ) the amount o f agonist high a f f i n i t y s t a t e (Rn) o f the receptor induced by an agonist and the r a t i o o f K . / K m were found t o c o r r e l a t e c l o s e l y wtih the i n t r i n s i c actiOity o f the agonist i n s t i m u l a t i n g adenylate c y c l a s e . In the alpha^-adrenergic receptor system o f the human p l a t e l e t s , R d i d not c o r r e l a t e with the i n t r i n s i c a c t i v i t y o f agonists f o r t h e i r a b i l i t y t o attenuate adenylate c y c l a s e s t i m u l a t i o n ( 3 6 ) . I n t h a t system, only the r a t i o o f K . / K appeared t o c o r r e l a t e with the i n t r i n s i c a c t i v i t y o f the alpha-adrenergic a g o n i s t s . Here, i n a n t e r i o r p i t u i t a r y membranes constant proportions o f both receptor s t a t e s were evidenced but r a t i o s of 3 0 - 2 0 0 were found f o r K , / K for a s e r i e s o f dopaminergic agonists despite the f a c t t h a t these agonists a l l appear t o display f u l l i n t r i n s i c a c t i v i t y in their a b i l i t y to inhibit p r o l a c t i n s e c r e t i o n from p i t u i t a r y c e l l s . In a d d i t i o n t o the e f f e c t s o f guanine n u c l e o t i d e s on the d i r e c t binding o f agonists and a n t a g o n i s t s , several f a c t o r s p o i n t toward the i n t e r a c t i o n of a p u t a t i v e n u c l e o t i d e binding p r o t e i n i n the mechanism of l i g a n d binding t o the dopamine receptor of a n t e r i o r p i t u i t a r y gland. F i r s t , both heat and NEM treatment of membranes mimic the q u a l i t a t i v e and q u a n t i t a t i v e M

M

M



90

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e f f e c t s of n u c l e o t i d e s on the binding of agonists and a n t a g o n i s t s . These treatments have been shown i n other systems to i n a c t i v a t e guanine n u c l e o t i d e r e g u l a t o r y p r o t e i n f u n c t i o n . Second, upon s o l u b i l i z a t i o n the a b i l i t y of the receptor to i n t e r a c t with high a f f i n i t y with agonists i s l o s t . However, l a b e l l i n g of the receptor i n membranes with the agonist [ H]n-propylapomorphine p r i o r to s o l u b i l i z a t i o n produces a s t a b l e a g o n i s t - r e c e p t o r complex s e n s i t i v e to guanine n u c l e o t i d e s . These data s t r o n g l y suggest the i n t e r a c t i o n of a guanine n u c l e o t i d e r e g u l a t o r y p r o t e i n i n the formation or s t a b i l i z a t i o n of the agonist high a f f i n i t y form of the receptor. We envisage t h a t the f o r m u l a t i o n proposed here of the same receptor s i t e e x i s t i n g i n two d i s t i n c t a f f i n i t y forms could p o s s i b l y represent the same e n t i t y as the Dp and D- subtypes of receptors proposed by Seeman ( 8 ) . However, much more work on the biochemical c h a r a c t e r i z a t i o n of the receptors from both p i t u i t a r y gland and the b r a i n w i l l be necessary before a f i r m conclusion can be reached. The dopamine receptor i n the p i t u i t a r y gland has the pharmacological c h a r a c t e r i s t i c s of the D« subtype of receptor. Recently, dopamine has been found to decrease c y c l i c AMP accumulation or i n h i b i t adenylate c y c l a s e i n the intermediate lobe and the a n t e r i o r p i t u i t a r y gland (2^,4,10,11,35). Thus, t h i s dopamine receptor may be l i n k e d physToTogiciTly to an i n h i b i t i o n of adenylate c y c l a s e s i m i l a r l y to the alpha^adrenergic receptor. Therefore, the high a f f i n i t y i n t e r a c t i o n of the receptor with agonists and i t s modulation by guanine n u c l e o t i d e s must be i m p l i c a t e d i n the biochemical t r a n s l a t i o n of agonist occupancy of the receptor to the e f f e c t o r system, adenylate c y c l a s e . I t has been suggested (IS) that a putative n u c l e o t i d e binding p r o t e i n d i f f e r e n t from that i m p l i c a t e d i n hormone s t i m u l a t i o n of adenylate c y c l a s e may be involved with a t t e n u a t i n g systems. Further s t u d i e s w i l l be r e q u i r e d , however, i n an attempt to demonstrate a d i r e c t p h y s i c a l i n t e r a c t i o n of the receptor with such a guanine n u c l e o t i d e binding p r o t e i n and i t s r e l a t i o n s h i p to that i m p l i c a t e d i n s t i m u l a t o r y systems. Literature Cited 1. Kebabian, J.W.; P e t z o l d , G.L.; Greengard, P. Proc. N a t l . Acad. Sci. USA 1972, 69, 2145-2149. 2. Cote, T.E.; Grewe, G.W.; Kebabian, J.W. Endocrinology 1982, 110, 805-811. 3. Meunier, H.; L a b r i e , F. L i f e Sci. 1982, 30, 963-968. 4. G i a n n a t t a s i o , G.; D e F e r r a r i , M.E.; Spada, A. L i f e Sci. 1981, 28, 1605-1612.


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Kebabian, J.W.; Calne, D.B. Nature 1979, 277, 93-96. T i t e l e r , M . ; List, S.; Seeman, P. Psychopharmacology 1979, 3, 411-420. T i t e l e r , M . ; Seeman, P. Eur. J . Pharmacol. 1979, 56, 291-292. Seeman, P. Pharmacol. Rev. 1980, 32, 229-313. Caron, M.G.; Beaulieu, M . ; Raymond, J.; Gagne, B . ; Drouin, J.; Lefkowitz, R.J.; Labrie, F. J. B i o l . Chem. 1978, 253, 2244-2253. Munenura, M . ; Cote, T . E . ; Tsuruta, K . ; Eskay, R . L . ; Kebabian, J.W. Endocrinology 1980, 106, 1676-1683. Labrie, F . ; Borgeat, T.; Barden, N . ; Godbout, M . ; Beaulieu, M . ; Ferland, L. "Polypeptide Hormones" (Beers, R.J., J r . and Bassett, E . G . , eds.); Raven Press: New York, 1980; pp. 235-251. De Lean, Α.; K i l p a t r i c k , B . F . ; Caron, M.G. Mol. Pharmacol. 1982, 22, 290-297. K i l p a t r i c k , B . F . ; De Lean, Α.; Caron, M.G. Mol. Pharmacol. 1982, 22, 298-303. De Lean, Α.; Hancock, Α.Α.; Lefkowitz, R . J . Mol. Pharmacol. 1982, 21, 5-16. Rodbell, M. Nature 1980, 284, 17-22. Rodbell, M . ; Krans, H.M.J.; Pohl, S . L . ; Birnbaumer, L. J. B i o l . Chem. 1971, 246, 1872-1876. Lefkowitz, R.J.; M u l l i k i n , D.; Caron, M.G. J. B i o l . Chem. 1976, 251, 4686-4692. De Lean, Α.; Stadel, J . M . ; Lefkowitz, R . J . J. B i o l . Chem. 1980, 255, 7108-7117. Stadel, J . M . ; Shorr, R.G.L.; Limbird, L.E.; Lefkowitz, R.J. J . B i o l . Chem. 1981, 256, 8718-8723. Kent, R . S . ; De Lean, Α.; Lefkowitz, R . J . Mol. Pharmacol. 1980, 17, 14-23. Stadel, J . M . ; De Lean, Α.; Lefkowitz, R . J . Adv. Enzymol. 1982, 53, 1-43. Ehlert, F.J.; Roeske, W.R.; Yamamura, H . I . J. Supramol. Struct. 1980, 14, 149-155. Burgisser, E . ; De Lean, Α.; Lefkowitz, R . J . Proc. Natl. Acad. S c i . USA 1982, 79, 1732-1736. Wolfe, B . B . ; Harden, T.K. J. Cyclic Nucleotide Res. 1981, 7, 303-312. De Lean, Α.; K i l p a t r i c k , B . F . ; Caron, M.G. Endocrinology 1982, 1064-1066. Stadel, J . M . ; Lefkowitz, R . J . Mol. Pharmacol. 1979, 16, 709-718. Ross, E.M.; Howlett, A . C . ; Ferguson, K.M.; Gilman, A.G. Biol. Chem. 1978, 253, 6406-6412. Gorissen, H . ; Laduron, P. Nature 1979, 279, 72-74. Davis, Α.; Madras, B . K . ; Seeman, P. Eur. J. Pharmacol. 1981, 70, 321-323.


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Commentary: The Dopamine Receptor of the Anterior Pituitary Gland S U S A N R. G E O R G E , M A S A Y U K I W A T A N A B E , and P H I L I P S E E M A N University of Toronto, Department of Pharmacology, Toronto, Canada

There is much evidence to suggest the presence of m u l t i p l e dopamine receptor subtypes (1). Of these subtypes, the a n t e r i o r p i t u i t a r y (AP) contains a pure population of postsynaptic D2 dopamine r e c e p t o r s , so that u n l i k e s t u d i e s i n b r a i n it is p o s s i b l e in AP to directly c o r r e l a t e receptor binding parameters to f u n c t i o n a l , p h y s i o l o g i c a l dopaminergic events such as the inhi bition of p r o l a c t i n s e c r e t i o n and the a t t e n u a t i o n of adenylate cyclase a c t i v i t y . The D2 receptor appears to e x i s t i n two s t a t e s , one with high and the other lower affinity f o r dopamine agonists. Both forms appear to have h i g h - a f f i n i t y f o r dopamine antagonists. In general, agonist competition curves o f 3H-agonist binding are monophasic with high Hill c o e f f i c i e n t s (~1), and agonist competi t i o n curves of 3H-antagonist binding are b i p h a s i c i n AP with low H i l l c o e f f i c i e n t s (

The Dopamine Receptor of the Anterior Pituitary Gland - ACS

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